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UV-VISIBLE SPECTROSCOPY
PRESENTED BY: ALEXA JACOB
M.pharm 1st yr
St.joseph’s college of
pharmacy,cherthala
1
 Electromagnetic radiation
Energy of an EMR
Electromagnetic spectrum & it’s regions
 Spectroscopy & it’s types
 Colorimetry
UV-spectroscopy
 Electronic transitions
Terms used in UV-Visible spectroscopy
Absorption band & it’s types
Beer lambert’s law & it’s deviations
⓫ Reference
2
 Electromagnetic radiation is a type of energy that is
transmitted through space at enormous velocities.
 Radiant energy has wave nature and being associated
with electric as well as magnetic field,these radiations
are called electromagnetic radiation.
 Electromagnetic radiation has it’s origin in atomic and
molecular processes.
3
 The field may be represented as electric and magnetic
vectors oscillating in mutually perpendicular planes.
4
 The energy of an EMR can be given by the following
equation:
Where E= Energy of radiation
h=Planck’s constant
µ=Frequency of radiation
E=hµ
5
Frequency(µ)= c/λ
Where c=velocity of light in vacuum
λ= wavelength
:-Hence, E=hµ
Therefore,energy of a radiation depends upon frequency
and wavelength of radiation.
E=hc/λ
6
The arrangement obtained by arranging various types
of electromagnetic waves or radiations in order of their
increasing wavelegth or decreasing frequencies is
called electromagnetic spectrum.
The electromagnetic spectrum is divided into a
number of regions; these are artificial divisions in the
sense that they have been defined solely as a result of
differences in the instrumentation required for
producing and detecting radiation of a given frequency
range.
7
8
 Ultraviolet: 190~400nm
 Violet: 400 - 420 nm
 Indigo: 420 - 440 nm
 Blue: 440 - 490 nm
 Green: 490 - 570 nm
 Yellow: 570 - 585 nm
 Orange: 585 - 620 nm
 Red: 620 - 780 nm
9
spectroscopy
10
Spectroscopy is the measurement and
interpretation of electromagnetic radiation
absorbed or emitted when the molecules or
atoms or ions of a sample moves from one
energy state to another energy state
SPECTROSCOPY
11
1) Atomic spectroscopy :
Here,the changes in energy takes
place at atomic level.
Eg: Atomic absorption spectroscopy, Flame
photometry
2)Molecular spectroscopy :
Here,the changes in energy takes
place at molecular level.
Eg: UV spectroscopy,colorimetry,infra red
spectroscopy
12
 Absorption spectrophotometry can be
defined as the measurement of absorption of radiant
energy by various substances.It involves the
measurement of absorptive capacity for radiant energy
in the visible,UV and IR regions of the spectrum.
13
o λ- 400-800nm
o Coloured substance absorbs light of different λ in
different manner and hence get an absorption
curve
o The λ at which maximum absorption takes place is
called as λmax
o λmax is characteristic for every coloured substance
o On plotting a graph of concentration v/s
absorbance,we get a calibration curve that is useful
in determining the concentration or amount of a
drug substance in the given sample solution. 14
15
 UV spectroscopy is concerned with the study of absorption
of uv radiation which ranges from 200-400nm.
 Valence electrons absorb the energy thereby molecules
undergoes transition from ground state to excited
state.
 This absorption is characteristic and depends on the
nature of electrons present.
 Types of electrons
σ electrons: in saturated compounds
π electrons: in unsaturated compounds
n electrons: in non bonded electrons
16
17
 σ electron from orbital is excited to corresponding
anti-bonding orbital σ*.
 The energy required is large for this transition.
 The organic compounds in which all the valence shell
electrons are involved in the formation of σ bond do
not show absorption in normal uv region (200-400nm)
 This transition is observed with saturated compounds.
1) σ-σ*
18
 Eg: Methane(CH₄) has C-H bond only and can undergo σ-
σ* transition and shows absorption maxima at 122nm.
 The usual spectroscopic technique cannot be used below
200 nm.
 To study this high energy transition,the entire region
should be evacuated (Vacuum uv region)
 Here,the excitation ocuurs with net retention of electronic
spin
 This region is less informative
19
 π electron in a bonding orbital is excited to
corresponding anti-bonding orbital π*.
 Energy required is less when compared to n-σ*
 Compounds containing multiple bonds like
alkenes,alkynes,carbonyls,nitriles,aromatic
compounds etc undergo π-π* transition.
Eg:Alkenes generally absorb in the region 170-205nm.
2) π-π*
20
 Absorption usually occurs in the ordinary uv
spectrophotometer
 Absorption bands in unconjugated alkenes (170-
190nm)
 Absorption bands in carbonyls (180 nm)
 Introduction of alkyl group in olefinic linkage
produces bathochromic shift
21
 Saturated compounds containing one hetero atom
with unshared pair of electrons(n) like O,N,S and
halogens are capable of n-σ* transition.
 These transition require less energy than σ-σ*
transition.
 In saturated alkyl halides, the energy required for
transition decrease with increase in the size of halogen
atom (or decrease in electronegativity)
3) n-σ*
22
 Eg:Methyl chloride has a λmax of 173nm.
Methyl iodide has a λmax of 258nm.
 This type of transition is very sensitive to hydrogen
bonding
Eg: Alcohol & amines
 Hydrogen bonding shift the uv absorptions to
shorter wavelength.
23
 An electron from non-bonding orbital is promoted to
anti-bonding π* orbital.
 Compounds containing double bonds involving
hetero atoms(C=O,N=O) undergo such type of
transitions.
 This transition require minimum energy out of all
transitions and shows absorption band at longer
wavelength around 300nm.
4) n-π*
24
 Eg:Saturated aldehydes shows both type of transitions
(n-π*, π-π*) at {low energy and high energy}
around 290 and 180 nm.
25
TERMS USED IN UV-
VISIBLE SPECTROSCOPY
26
 Chromophore is defined as the nucleus or any
isolated covalently bonded group responsible for the
absorption of light radiation.
 Any group which exhibits absorption of
electromagnetic radiations in the visible or ultraviolet
region.
C=C , C=O ,NO2 etc
 Some of the important chromophores are
carbonyls,acids,esters,nitrile,ethylenic groups.
27
28
 These are co-ordinatively saturated or un-saturated
groups which themselves do not absorb radiations,but
when present alongwith a chromophore enhances the
absorbing properties of chromophore.
 Also known as colour enhancing group.
 All auxochromes have one or more non-bonding pair
of electrons.
-NH2 ,-OH ,-OR,-COOH etc
 It extend the conjugation of a chromophore by sharing
the non-bonding electrons. 29
30
ABSORPTION &
INTENSITY SHIFTS
31
 When the absorption maxima(λmax)of a compound
shifts to longer wavelength,it is known as
bathochromic shift or red shift.
 The effect is due to the presence of auxochrome or by
change of solvent.
 Eg: The n-π* transition for carbonyl compounds
experiences bathochromic shift when the polarity of
solvent is decreased.
1) Bathochromic shift(red shift)
32
 When the absorption maxima (λmax) of a compound
shifts to a shorter wavelength,it is known as
hypsochromic shift or blue shift.
 The effect is due to the presence of a group causes
removal of conjugation or by change of solvent.
2) Hypsochromic shift(blue shift)
33
Eg:
Aniline shows blue shift in acidic medium since it loses
conjugation. Aniline(280nm) & Anilinium ion (-
203nm).
34
3) Hyperchromic effect
35
4) Hypochromic effect
36
SHIFTS & EFFECTS
37
ABSORPTION
BANDS
38
 The spectrum consist of sharp peaks and each peak will
correspond to the promotion of electron from one
electronic level to another.
 During promotion,the electron moves from a given
vibrational and rotational level within one electronic mode
to the other within the next electronic mode.
 Thus,there will be a large no of possible transitions
 Hence,not just one but a large no. of wavelengths which
are close enough will be absorbed resulting in the
formation of bands 39
1) K –Band
 K-Bands originate due to π-π* transition from a
compound containing a conjugated system
Such type of bands arise in compounds like
dienes,polyenes and enones etc.
Compound Transition λmax(nm) εmax
Acetophenone π-π* 240 13,000
1,3-butadiene π-π* 217 21,000
40
R-Band transition originate due to n-π* transition of a
single chromophoric group and having atleast one
lone pair of electrons on the hetero atom
These are less intense with εmax value below 100
Compound Transition λmax(nm) εmax
Acetone n-π* 270 15
Acetaldehyde n-π* 293 12
41
Such type of bands arise due to π-π* transition in
aromatic or hetero-aromatic molecules.
Benzene shows absorption peaks between 230-
270nm.when a chromophoric group is attached to the
benzene ring ,the B-Bands are observed at longer
wavelengths than the more intense K-Bands.
Compound Transition λmax(nm) εmax
Benzene π-π* 255 215
Phenol π- π* 270 1450
42
E-Band originate due to the electronic transitions in
the benzenoid systems of three ethylenic bonds which
are in closed cyclic conjugation.
These are further characterized as E1 and E2 bands
E1 band which appear at shorter wavelength is usually
more intense than the E2 band for the same
compound which appears at longer wavelength.
Compound E1 Band E1 Band E2 Band E2 Band
λmax(nm) εmax λmax(nm) εmax
Benzene 184 50,000 204 79,000
Napthalene 221 133,000 286 9,300
43
44
BEER’S LAW
According to this law,when a beam of monochromatic
radiation is passed through a solution of absorbing species,the
intensity of beam of monochromatic light decreases exponentially
with increase in concentration of absorbing species
LAMBERT’S LAW
Lambert’s law states that the rate of decrease of
intensity of monochromatic light with the thickness of the
medium is directly proportional to the intensity of incident
light.
-dI/dc α I
-dI/dt α I
45
According to beer’s law,
-dI α I
dc
The decrease in the intensity of light (I) with
concentration(c) is proportional to intensity of
incident light(I)
-dI = K.I { removing & introducing the
dc constant of proportionality
“K”}
-dI = K.dc { rearranging terms}
I
46
On Integrating the equation,
-∫dI = K.∫dc
I
-ln I = K.c + b  { b constant of integration}
When concentration =0, there is no absorbance,Hence I=I₀
Substituting in equation 
-ln I₀ = K*0+b
-ln I₀ = b
Substituting the value of b in equation
-ln I = K.c-ln I₀
ln I₀ - ln I = Kc { since log A-logB = log A }
B
ln I₀ = Kc
I
47
I₀ = ekc { removing natural log }
I
I = e-kc {making inverse on both sides}
I₀
I = I₀e-kc  [equation for beer’s law]
According to lambert’s law,
-dI α I
dt
This eqn can be simplified by replacing ‘c’ with ‘t’ in
I = I₀e-kt 
Eqn  &  can be combined to get I = I₀e-kct 48
Converting natural log to base 10 & K= k x 0.4343
I = ₁₀-kct { rearranging terms }
I₀
I₀ = ₁₀kct { inverse on both sides}
I
Taking log on both sides ,
log I₀ = Kct 
I
Here, Transmittance T = I₀ , Absorbance,A = log 1
I T
49
A = log 1
T
A = log 1 { Since T = I₀ }
I/I₀ I
A =log I₀ 
I
Using eqn  & , since A =log I₀ and log I₀ = Kct ,
I I
A= Kct
Instead of K, we can use ε
A = εct { Mathematical eqn for beer lambert’s
law}
50
51
52
There are 3 types of deviations usually observed
A)The real limitation of the law is that the beer’s law is
successful in describing the absorption behaviour of
dilute solutions only.
B)CHEMICAL DEVIATIONS:
Association of molecules
This can be explained by taking the examples of
methylene blue at small concentration(10‾⁵ molar) and
at concentration above 10‾⁵molar.
53
 Dissociation of molecules
This can be explained by the fact that dichromate
ions posses their maximum absorbance at 450nm
which is orange in colour .But upon dilution,it will be
dissociated to chromate ions having maximum
absorbance at 410nm which is yellow in colour.
 This law is not valid in case if the absorbing material
is coagulated into a small number of large units.
This law shows deviation if the absorbing material at
the required wavelength contains presence of
impurities.
54
 This law is not applicable in case of suspension.
C)INSTRUMENTAL DEVIATIONS:
 Strict adherence of an absorbing system to this law is
observed only when the radiation used is monochromatic.
Stray radiation,slit width also causes deviation.
Hence,the reasons for the deviation depends on
environment such as temperature,pressure,solvent,
refractive index of the sample
55
 Absorbance
0.800
λ=430nm
0.600 λ=570nm
0.400
0.200
0.00
0.00 4.00 8.00 12.00 16.00 concentration
56
 Absorbance
0.800 ε=1000 ε=1500
0.600
ε=1750
0.400
0.200
0.00 2.0 4.0 6.0 8.0 10.0 concentration
57
Absorbance 0.0%
 2.0 0.2%
 1%
 1.0 5%
 0
 2.5 5.0 7.5 10 concentration
58
1)Elementary organic spectroscopy,principles & chemical
applications,Y.R Sharma,Revised edition,pg n.o
18,26,27
2) Pharmaceutical chemistry,Instrumental
techniques,vol 2,Leslie.G.chatten,pg n.o 21-24
3)Principles and practice of analytical chemistry,F.W
Fifield & D.kealey, 5th edition ,pg n.o 270-274
4)Pharmaceutical analysis,P.Parimoo, pg n.o
147,151,152,165
5)Industrial methods of chemical analysis,B.K
Sharma,pg n.o 46-65,91-113
59
 6)Instrumental analysis,Skoog,Holler,Crouch, pg
no.383,386
 7)Practical pharmaceutical chemistry,4th edition,partv
2, Beckett ,stenlake, pg n.o275-277
60


THANKYOU…
61

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Uv visible spectroscopy ppt

  • 1. UV-VISIBLE SPECTROSCOPY PRESENTED BY: ALEXA JACOB M.pharm 1st yr St.joseph’s college of pharmacy,cherthala 1
  • 2.  Electromagnetic radiation Energy of an EMR Electromagnetic spectrum & it’s regions  Spectroscopy & it’s types  Colorimetry UV-spectroscopy  Electronic transitions Terms used in UV-Visible spectroscopy Absorption band & it’s types Beer lambert’s law & it’s deviations ⓫ Reference 2
  • 3.  Electromagnetic radiation is a type of energy that is transmitted through space at enormous velocities.  Radiant energy has wave nature and being associated with electric as well as magnetic field,these radiations are called electromagnetic radiation.  Electromagnetic radiation has it’s origin in atomic and molecular processes. 3
  • 4.  The field may be represented as electric and magnetic vectors oscillating in mutually perpendicular planes. 4
  • 5.  The energy of an EMR can be given by the following equation: Where E= Energy of radiation h=Planck’s constant µ=Frequency of radiation E=hµ 5
  • 6. Frequency(µ)= c/λ Where c=velocity of light in vacuum λ= wavelength :-Hence, E=hµ Therefore,energy of a radiation depends upon frequency and wavelength of radiation. E=hc/λ 6
  • 7. The arrangement obtained by arranging various types of electromagnetic waves or radiations in order of their increasing wavelegth or decreasing frequencies is called electromagnetic spectrum. The electromagnetic spectrum is divided into a number of regions; these are artificial divisions in the sense that they have been defined solely as a result of differences in the instrumentation required for producing and detecting radiation of a given frequency range. 7
  • 8. 8
  • 9.  Ultraviolet: 190~400nm  Violet: 400 - 420 nm  Indigo: 420 - 440 nm  Blue: 440 - 490 nm  Green: 490 - 570 nm  Yellow: 570 - 585 nm  Orange: 585 - 620 nm  Red: 620 - 780 nm 9
  • 11. Spectroscopy is the measurement and interpretation of electromagnetic radiation absorbed or emitted when the molecules or atoms or ions of a sample moves from one energy state to another energy state SPECTROSCOPY 11
  • 12. 1) Atomic spectroscopy : Here,the changes in energy takes place at atomic level. Eg: Atomic absorption spectroscopy, Flame photometry 2)Molecular spectroscopy : Here,the changes in energy takes place at molecular level. Eg: UV spectroscopy,colorimetry,infra red spectroscopy 12
  • 13.  Absorption spectrophotometry can be defined as the measurement of absorption of radiant energy by various substances.It involves the measurement of absorptive capacity for radiant energy in the visible,UV and IR regions of the spectrum. 13
  • 14. o λ- 400-800nm o Coloured substance absorbs light of different λ in different manner and hence get an absorption curve o The λ at which maximum absorption takes place is called as λmax o λmax is characteristic for every coloured substance o On plotting a graph of concentration v/s absorbance,we get a calibration curve that is useful in determining the concentration or amount of a drug substance in the given sample solution. 14
  • 15. 15
  • 16.  UV spectroscopy is concerned with the study of absorption of uv radiation which ranges from 200-400nm.  Valence electrons absorb the energy thereby molecules undergoes transition from ground state to excited state.  This absorption is characteristic and depends on the nature of electrons present.  Types of electrons σ electrons: in saturated compounds π electrons: in unsaturated compounds n electrons: in non bonded electrons 16
  • 17. 17
  • 18.  σ electron from orbital is excited to corresponding anti-bonding orbital σ*.  The energy required is large for this transition.  The organic compounds in which all the valence shell electrons are involved in the formation of σ bond do not show absorption in normal uv region (200-400nm)  This transition is observed with saturated compounds. 1) σ-σ* 18
  • 19.  Eg: Methane(CH₄) has C-H bond only and can undergo σ- σ* transition and shows absorption maxima at 122nm.  The usual spectroscopic technique cannot be used below 200 nm.  To study this high energy transition,the entire region should be evacuated (Vacuum uv region)  Here,the excitation ocuurs with net retention of electronic spin  This region is less informative 19
  • 20.  π electron in a bonding orbital is excited to corresponding anti-bonding orbital π*.  Energy required is less when compared to n-σ*  Compounds containing multiple bonds like alkenes,alkynes,carbonyls,nitriles,aromatic compounds etc undergo π-π* transition. Eg:Alkenes generally absorb in the region 170-205nm. 2) π-π* 20
  • 21.  Absorption usually occurs in the ordinary uv spectrophotometer  Absorption bands in unconjugated alkenes (170- 190nm)  Absorption bands in carbonyls (180 nm)  Introduction of alkyl group in olefinic linkage produces bathochromic shift 21
  • 22.  Saturated compounds containing one hetero atom with unshared pair of electrons(n) like O,N,S and halogens are capable of n-σ* transition.  These transition require less energy than σ-σ* transition.  In saturated alkyl halides, the energy required for transition decrease with increase in the size of halogen atom (or decrease in electronegativity) 3) n-σ* 22
  • 23.  Eg:Methyl chloride has a λmax of 173nm. Methyl iodide has a λmax of 258nm.  This type of transition is very sensitive to hydrogen bonding Eg: Alcohol & amines  Hydrogen bonding shift the uv absorptions to shorter wavelength. 23
  • 24.  An electron from non-bonding orbital is promoted to anti-bonding π* orbital.  Compounds containing double bonds involving hetero atoms(C=O,N=O) undergo such type of transitions.  This transition require minimum energy out of all transitions and shows absorption band at longer wavelength around 300nm. 4) n-π* 24
  • 25.  Eg:Saturated aldehydes shows both type of transitions (n-π*, π-π*) at {low energy and high energy} around 290 and 180 nm. 25
  • 26. TERMS USED IN UV- VISIBLE SPECTROSCOPY 26
  • 27.  Chromophore is defined as the nucleus or any isolated covalently bonded group responsible for the absorption of light radiation.  Any group which exhibits absorption of electromagnetic radiations in the visible or ultraviolet region. C=C , C=O ,NO2 etc  Some of the important chromophores are carbonyls,acids,esters,nitrile,ethylenic groups. 27
  • 28. 28
  • 29.  These are co-ordinatively saturated or un-saturated groups which themselves do not absorb radiations,but when present alongwith a chromophore enhances the absorbing properties of chromophore.  Also known as colour enhancing group.  All auxochromes have one or more non-bonding pair of electrons. -NH2 ,-OH ,-OR,-COOH etc  It extend the conjugation of a chromophore by sharing the non-bonding electrons. 29
  • 30. 30
  • 32.  When the absorption maxima(λmax)of a compound shifts to longer wavelength,it is known as bathochromic shift or red shift.  The effect is due to the presence of auxochrome or by change of solvent.  Eg: The n-π* transition for carbonyl compounds experiences bathochromic shift when the polarity of solvent is decreased. 1) Bathochromic shift(red shift) 32
  • 33.  When the absorption maxima (λmax) of a compound shifts to a shorter wavelength,it is known as hypsochromic shift or blue shift.  The effect is due to the presence of a group causes removal of conjugation or by change of solvent. 2) Hypsochromic shift(blue shift) 33
  • 34. Eg: Aniline shows blue shift in acidic medium since it loses conjugation. Aniline(280nm) & Anilinium ion (- 203nm). 34
  • 39.  The spectrum consist of sharp peaks and each peak will correspond to the promotion of electron from one electronic level to another.  During promotion,the electron moves from a given vibrational and rotational level within one electronic mode to the other within the next electronic mode.  Thus,there will be a large no of possible transitions  Hence,not just one but a large no. of wavelengths which are close enough will be absorbed resulting in the formation of bands 39
  • 40. 1) K –Band  K-Bands originate due to π-π* transition from a compound containing a conjugated system Such type of bands arise in compounds like dienes,polyenes and enones etc. Compound Transition λmax(nm) εmax Acetophenone π-π* 240 13,000 1,3-butadiene π-π* 217 21,000 40
  • 41. R-Band transition originate due to n-π* transition of a single chromophoric group and having atleast one lone pair of electrons on the hetero atom These are less intense with εmax value below 100 Compound Transition λmax(nm) εmax Acetone n-π* 270 15 Acetaldehyde n-π* 293 12 41
  • 42. Such type of bands arise due to π-π* transition in aromatic or hetero-aromatic molecules. Benzene shows absorption peaks between 230- 270nm.when a chromophoric group is attached to the benzene ring ,the B-Bands are observed at longer wavelengths than the more intense K-Bands. Compound Transition λmax(nm) εmax Benzene π-π* 255 215 Phenol π- π* 270 1450 42
  • 43. E-Band originate due to the electronic transitions in the benzenoid systems of three ethylenic bonds which are in closed cyclic conjugation. These are further characterized as E1 and E2 bands E1 band which appear at shorter wavelength is usually more intense than the E2 band for the same compound which appears at longer wavelength. Compound E1 Band E1 Band E2 Band E2 Band λmax(nm) εmax λmax(nm) εmax Benzene 184 50,000 204 79,000 Napthalene 221 133,000 286 9,300 43
  • 44. 44
  • 45. BEER’S LAW According to this law,when a beam of monochromatic radiation is passed through a solution of absorbing species,the intensity of beam of monochromatic light decreases exponentially with increase in concentration of absorbing species LAMBERT’S LAW Lambert’s law states that the rate of decrease of intensity of monochromatic light with the thickness of the medium is directly proportional to the intensity of incident light. -dI/dc α I -dI/dt α I 45
  • 46. According to beer’s law, -dI α I dc The decrease in the intensity of light (I) with concentration(c) is proportional to intensity of incident light(I) -dI = K.I { removing & introducing the dc constant of proportionality “K”} -dI = K.dc { rearranging terms} I 46
  • 47. On Integrating the equation, -∫dI = K.∫dc I -ln I = K.c + b  { b constant of integration} When concentration =0, there is no absorbance,Hence I=I₀ Substituting in equation  -ln I₀ = K*0+b -ln I₀ = b Substituting the value of b in equation -ln I = K.c-ln I₀ ln I₀ - ln I = Kc { since log A-logB = log A } B ln I₀ = Kc I 47
  • 48. I₀ = ekc { removing natural log } I I = e-kc {making inverse on both sides} I₀ I = I₀e-kc  [equation for beer’s law] According to lambert’s law, -dI α I dt This eqn can be simplified by replacing ‘c’ with ‘t’ in I = I₀e-kt  Eqn  &  can be combined to get I = I₀e-kct 48
  • 49. Converting natural log to base 10 & K= k x 0.4343 I = ₁₀-kct { rearranging terms } I₀ I₀ = ₁₀kct { inverse on both sides} I Taking log on both sides , log I₀ = Kct  I Here, Transmittance T = I₀ , Absorbance,A = log 1 I T 49
  • 50. A = log 1 T A = log 1 { Since T = I₀ } I/I₀ I A =log I₀  I Using eqn  & , since A =log I₀ and log I₀ = Kct , I I A= Kct Instead of K, we can use ε A = εct { Mathematical eqn for beer lambert’s law} 50
  • 51. 51
  • 52. 52
  • 53. There are 3 types of deviations usually observed A)The real limitation of the law is that the beer’s law is successful in describing the absorption behaviour of dilute solutions only. B)CHEMICAL DEVIATIONS: Association of molecules This can be explained by taking the examples of methylene blue at small concentration(10‾⁵ molar) and at concentration above 10‾⁵molar. 53
  • 54.  Dissociation of molecules This can be explained by the fact that dichromate ions posses their maximum absorbance at 450nm which is orange in colour .But upon dilution,it will be dissociated to chromate ions having maximum absorbance at 410nm which is yellow in colour.  This law is not valid in case if the absorbing material is coagulated into a small number of large units. This law shows deviation if the absorbing material at the required wavelength contains presence of impurities. 54
  • 55.  This law is not applicable in case of suspension. C)INSTRUMENTAL DEVIATIONS:  Strict adherence of an absorbing system to this law is observed only when the radiation used is monochromatic. Stray radiation,slit width also causes deviation. Hence,the reasons for the deviation depends on environment such as temperature,pressure,solvent, refractive index of the sample 55
  • 57.  Absorbance 0.800 ε=1000 ε=1500 0.600 ε=1750 0.400 0.200 0.00 2.0 4.0 6.0 8.0 10.0 concentration 57
  • 58. Absorbance 0.0%  2.0 0.2%  1%  1.0 5%  0  2.5 5.0 7.5 10 concentration 58
  • 59. 1)Elementary organic spectroscopy,principles & chemical applications,Y.R Sharma,Revised edition,pg n.o 18,26,27 2) Pharmaceutical chemistry,Instrumental techniques,vol 2,Leslie.G.chatten,pg n.o 21-24 3)Principles and practice of analytical chemistry,F.W Fifield & D.kealey, 5th edition ,pg n.o 270-274 4)Pharmaceutical analysis,P.Parimoo, pg n.o 147,151,152,165 5)Industrial methods of chemical analysis,B.K Sharma,pg n.o 46-65,91-113 59
  • 60.  6)Instrumental analysis,Skoog,Holler,Crouch, pg no.383,386  7)Practical pharmaceutical chemistry,4th edition,partv 2, Beckett ,stenlake, pg n.o275-277 60